The objective of this proposal is two-fold; to construct a gene-fusion between the ribosomal RNA promoter (rrnP) and some suitable well characterized operon such as tryptophan (trp) and to use the rrnP-fusion to study various aspects of rRNA genetics and control. The genetic fusion of two different operons (for example, trp-lac, and various lambda-trp fusions) has provided an added dimension to the selective techniques available to the Escherichia coli geneticist and has provided a facile means of observing the regulation of genes specifying products which are not readily measured. A specialized transducing phi80d3 which carries one copy of the E. coli rRNA operon (rrn) has been isolated. We intend to construct the desired gene fusion in vitro using restriction endonucleases. The recent purification of restriction endonucleases and characterization of the DNA sequences they recognize has made possible the assembly of genetic information from a variety of sources in a highly controlled fashion. Cleavage of phi80d3 DNA with RI endonuclease gives rise to unique DNA fragments. An RI recognition sequence within the 16S rrn gene has been revealed by the nucleotide sequence work of Fellner. Therefore one of the phi80d3 fragments must carry rrnP followed by about 750 base pairs. This fragment and a suitable trp RI restriction fragment will be inserted into the self-replicating plasmid colEl. Once obtained, the rrnP-fusion will be used to study various aspects of rRNA genetics and control, such as the mechanism of rRNA transcription initiation and control. In addition, the fusions will be used to select mutants at rRNA control sites which will subsequently be studied both in vivo and in vitro. The promoter fusion and these mutants will be used to locate and determine the nucleotide sequences of the control elements of the rrn operon.